CD4+T cells play a critical role in the induction and maintenance of anti-tumor responses. In order to develop and optimize immunotherapeutic approaches to stimulate human tumor antigen (TA)-specific CD4+ T cells in vivo, we have previously identified a number of promiscuous MHC class II-restricted epitopes from cancer- germline antigens (CGAs), melanocyte-lineage antigens (MDAs) and overexpressed or universal antigens. We have shown that these epitopes stimulate spontaneous circulating TA-specific CD4+ T cells in peripheral blood lymphocytes (PBLs) of patients with advanced melanoma. Most recently, we have shown that TA-specific CD4+ T cells were either T helper or regulatory T cells (T regs). The goal of this proposal is to expand the functional studies of TA-specific regs isolated from patients with advanced melanoma. This research proposal stems from novel findings that be can be stated as follows: 1) MHC class II epitopes stimulate not only spontaneous Foxp3- T-helper and Foxp3+ T regs but also Foxp3+ T helper and Foxp3- T regs, questioning the reliability of Foxp3 as a marker of T regs in humans; 2) in contrast to naturally-occurring CD4+CD25+T regs, TA-specific T regs inhibit TA cross-presentation by DCs and cross-priming of TA-specific CD8+ T cells; 3) TA- specific T regs act on autologous B cells to inhibit IgG antibody production and stimulate IL-10 production; 4) Immature monocyte-derived DCs (iDCs) generated in an immunosuppressive milieu (IL-10 and TGF-2) can be activated by tumor antigen/antibody immune complexes (TA/Ab IC), promoting TA cross-presentation to CD8+ T cells and CD4+ T-helper functions. Collectively, the data generated in this proposal will develop the knowledge required for the induction of potent TA-specific T-helper responses in vivo in patients with advanced melanoma. They will provide a better understanding of the mechanisms by which TA-specific T regs inhibit cross-presentation/cross-priming by DCs and B cell antibody production. Therefore, they will contribute to the development of strategies aimed at expanding immune responses integrating TA-specific T helper cells, TA- specific CD8+ T cells and antibody responses in patients with melanoma. PUBLIC HEALTH RELEVANCE: The proposed study aims at understanding how regulatory T cells recognizing small fragments of protein specifically expressed by melanoma cells (i.e. epitopes), inhibit immune responses to melanoma. This research project will develop the knowledge required for the implementation of novel active and specific immunotherapies to stimulate potent anti-tumor responses in melanoma patients.